Muscarinic antagonists with PARP and SIR modulating activity as agents for inflammatory diseases

ABSTRACT

The present invention relates generally to the cytoprotective activity of mixed muscarinic inhibition/PARP modulation and in particular to the use of dual inhibitors of M1 muscarinic receptor and poly(ADP-ribose) polymerase (PARP) as epithelioprotective medicaments, particularly as medicaments for the prevention and/or treatment of at least one of the common lung diseases associated with a significant inflammatory component such as severe sepsis, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, asthma, allergic rhinitis, chronic obstructive pulmonary disease, pulmonary fibrosis, systemic sclerosis, pneumoconiosis or lung cancer. Particularly preferred compounds are condensed diazepinones, e.g. condensed benzodiazepinones such as pirenzepine or compounds which are metabolized to condensed benzodiazepinones such as olanzapine.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC §371 National Phase Entry Application fromPCT/EP2005/007805, filed Jul. 18, 2005, and designating the UnitedStates, which claims the benefit of U.S. Provisional 60/588,354 filedJul. 16, 2004, 60/620,323 filed Oct. 21, 2004, 60/656,378 filed Feb. 28,2005, and 60/656,379 filed Feb. 28, 2005.

The present invention relates to generally to the cytoprotectiveactivity of mixed muscarinic inhibition/PARP modulation and/or SIR2modulation and in particular to the use of dual inhibitors of M1muscarinic receptor and poly (ADP-ribose) polymerase (PARP) and/ormodulators of SIR2 as epithelioprotective medicaments, particularly asmedicaments for the prevention and/or treatment of diseases associatedwith a significant inflammatory component, especially lung diseases suchas severe sepsis, acute lung injury, acute respiratory distresssyndrome, cystic fibrosis, asthma, allergic rhinitis, chronicobstructive pulmonary disease, pulmonary fibrosis, systemic sclerosis,pneumoconiosis or lung cancer. Particularly preferred compounds arecondensed diazepinones, e.g. condensed benzodiazepinones such aspirenzepine or compounds which are metabolized to condensedbenzodiazepinones such as olanzapine.

Pirenzepine(5,11-dihydro-11[(4-methyl-1-piperazinyl)-acetyl]-6H-pyrido-[2,3-b]-[1,4]benzodiazepine-6-one),is a topical antiulcerative M1 muscarinic antagonist, that inhibitsgastric secretion at lower doses than are required to affectgastrointestinal motility, salivary, central nervous system,cardiovascular, ocular, and urinary function. It promotes the healing ofduodenal ulcers and due to its cytoprotective action is beneficial inthe prevention of duodenal ulcer recurrence. It also potentiates theeffect of other antiulcer agents such as cimetidine and ranitidine. Itis generally well tolerated by patients. The M1 muscarinic effect ofpirenzepine is thought to be an explanation for this and a variety ofadditional effects in other indications, listed below.

For the preparation, pharmacology, pharmacokinetics and mechanism ofaction of pirenzepine, the following references are referred to:

-   Preparation: FR. Patent. 1,505,795 (1967 to Thomae), CA. 70, 4154w    (1969).-   Pharmacology: W. Ebenem et al. Arzneimittel-Forsch. 27, 356 (1977).-   Pharmacokinetics: R. Hammes et al., ibid. 928.-   Mechanism of action: G. Heller et al., Verh. Deut Ges. Inn. Med. 84,    991 (1978), C.A. 90, 132984s (1979).-   Human pharmacology: H. Brunnen et al., Arzneimittel-Forsch. 27, 684    (1977). Multicenter controlled clinical trial: Scand. J.    Gastroenterol. 17, Suppl. 81, 1-42 (1982). Review of pharmacology    and therapeutic efficacy: A. A. Carmine, R. N. Brogden, Drugs 30,    85-126 (1985).-   Comprehensive description: H. A. El-Obeid et al., in Analytical    Profiles of Drug Substances, Vol 16, K. Florey, Ed. (Academic Press,    New York, 1987) pp 445-506.

The M1 muscarinic effect of pirenzepine is thought to be responsible forvago-mimetic neuro-humoral regulation potentially useful for treatmentof chronic heart failure patients and of patients recovering frommyocardial infarction or generally in hypertension [Jakubetz J Humancardiac beta1- or beta2-adrenergic receptor stimulation and the negativechronotropic effect of low-dose pirenzepine. Clin Pharmacol Ther—2000May; 67(5): 549-57. Hayano T, Shimizu A, Ikeda Y, Yamamoto T, YamagataT, Ueyama T, Furutani Y, Matsuzaki M Paradoxical effects of pirenzepineon parasympathetic activity in chronic heart failure and control. Int.J. Cardiol. 1999 January; 68(1):47-56. Pedreffi R F, Colombo E, Braga SS, Ballardini L, Caru B Effects of oral pirenzepine on heart ratevariability and baroreceptor reflex sensitivity after acute myocardialinfarction. J. Am. Coll. Cardiol. 1995 Mar. 15; 25(4):915-21. WilhelmyR, Pitschner H, Neuzner J, Dursch M, Konig S Selective and unselectiveblockade of sympathicus and parasympathicus and vagal enhancement bypirenzepine: effects on heart rate and heart rate variability in healthysubjects. Clin Sci (Colch) 1996; 91 Suppl: 124.].

Pirenzepine has also been implicated in some CNS-related diseases basedon its M1 muscarinic inhibitory action, e.g. it is used as aco-medication to antipsychotic drugs (Hedges D, Jeppson K, Whitehead PAntipsychotic medication and seizures: a review. Drugs Today (Barc).2003 July; 39(7):551-7; Schneider B, Weigmann H, Hiemke C, Weber B,Fritze J. Reduction of clozapine-induced hypersalivation by pirenzepineis safe. Pharmacopsychiatry. 2004 March; 37(2):43-5). A potential roleof muscarinic receptors in schizophrenia is assumed to be the underlyingreason (Katerina Z, Andrew K, Filomena M, Xu-Feng H. Investigation ofm1/m4 muscarinic receptors in the anterior cingulate cortex inschizophrenia, bipolar disorder, and major depression disorder.Neuropsychopharmacology. 2004 Mar.; 29(3): 619-25). Also selectivemuscarinic M1 agonists have been implicated in the release andprocessing of amyloid precursor protein potentially relevant inAlzheimer's disease (Qiu Y, Wu X J, Chen H Z. Simultaneous changes insecretory amyloid precursor protein and beta-amyloid peptide releasefrom rat hippocampus by activation of muscarinic receptors. NeurosciLett. 2003 Nov. 27; 352(1):41-4; Qiu Y, Chen H Z, Wu X J, Jin Z J.6beta-acetoxy nortropane regulated processing of amyloid precursorprotein in CHOMI cells and rat brain. Eur J. Pharmacol. 2003 May 2;468(1):1-8.).

Pirenzepine is used together with drugs like olanzapine or clozapine tosuppress side effects (e.g. emesis or hypersalivation) in cancer orschizophrenia treatments (Bai Y M, Lin C C, Chen J Y, Liu W C.Therapeutic effect of pirenzepine for clozapine-induced hypersalivation:a randomized, double-blind, placebo-controlled, cross-over study. J ClinPsychopharmacol. 2001 Dec.; 21(6):608-11).

Pirenzepine has also been found to be effective in the reduction ofprogression of myopia, especially in children with promising efficacyresults and acceptable safety profile (Gilmartin B. Myopia: precedentsfor research in the twenty-first century. Clin Experiment Opthalmol.2004 June; 32(3):305-24; Bartlett J D, Niemann K, Houde B, Allred T,Edmondson M J, Crockett R S. A tolerability study of pirenzepineophthalmic gel in myopic children. J Ocul Pharmacol Ther. 2003 Jun.;19(3):271-9.).

Further, pirenzepine has been tested in the treatment of diabetes (IssaB G, Davies N, Hood K, Premawardhana L D, Peters J R, Scanlon M F.Effect of 2-week treatment with pirenzepine on fasting and postprandialglucose concentrations in individuals with type 2 diabetes. DiabetesCare. 2003 May; 26(5):1636-7). Taken together, these studies and othersshow that pirenzepine is a relatively safe compound.

There is no evidence for a neuroprotective or cytoprotective role ofmuscarinic receptors. Only their role in modulating potentiallyexcitotoxic glutamate release has been discussed (e.g. Sholl-Franco A,Marques P M, Ferreira C M, de Araujo E G. IL-4 increases GABAergicphenotype in rat retinal cell cultures: involvement of muscarinicreceptors and protein kinase C. J Neuroimmunol. 2002 December;133(1-2):20-9. Calabresi P, Picconi B, Saulle E, Centonze D, HainsworthA H, Bernardi G. Is pharmacological neuroprotection dependent on reducedglutamate release? Stroke. 2000 Mar.; 31(3):766-72; discussion 773).Muscarinic receptors modulate the mRNA expression of NMDA receptors inbrainstem and the release of glutamate. The central role of glutamatereceptors in mediating excitotoxic neuronal death in stroke, epilepsyand trauma has been well established. Although calcium ions areconsidered key regulators of excitotoxicity, new evidence suggests thatspecific second messenger pathways rather than total Ca²⁺ load areresponsible for mediating neuronal degeneration. Evidence exists showingthat inhibiting signals downstream of glutamate receptors, such asnitric oxide and PARP-1 can reduce excitotoxic insult. (Aarts M M,Tymianski M. Molecular mechanisms underlying specificity of excitotoxicsignaling in neurons. Curr Mol. Med. 2004 Mar.; 4(2):137-47).

Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damageand is catalyzed by poly(ADP-ribose) polymerase (PARP-1). Directlystimulated by DNA breaks, PARP-1 is involved in a variety ofphysiological and pathophysiological phenomena. Physiologically it isimportant for maintaining genomic stability. Pathophysiologically,PARP-1 overactivity contributes to a number of diseases associated withcellular stress. Proteolysis of PARP is, along with fragmentation ofDNA, one of the hallmarks of apoptosis. PARP, is a DNA damage sensorenzyme that normally functions in DNA repair, but promotes cell deathwhen extensively activated by DNA damage, which leads to celldysfunction and cell death mainly due to depletion of NAD⁺ (thesubstrate of PARP-1) and ATP. Overactivation of PARP appears to beprominent in vascular stroke and other neurodegenerative diseasescausing necrotic neural death. Therefore PARP inhibitors have drawnintense interest in the recent past as potential cyto-/neuroprotectivelead structures with a broad based therapeutic potential, in particularof PARP-1 inhibitors (e.g. Cosi C, Guerin K, Marien M, Koek W, Rollet K.The PARP inhibitor benzamide protects against kainate and NMDA but notAMPA lesioning of the mouse striatum in vivo. Brain Res. 2004 Jan. 16;996(1):1-8. Suh S W, Aoyama K, Chen Y, Garnier P, Matsumori Y, Gum E,Liu J, Swanson R A. Hypoglycemic neuronal death and cognitive impairmentare prevented by poly(ADP-ribose) polymerase inhibitors administeredafter hypoglycemia. J Neurosci. 2003. 23:10681-90. Pogrebniak A,Schemainda I, Pelka-Fleischer R, Nussler V, Hasmann M. Poly ADP-ribosepolymerase (PARP) inhibitors transiently protect leukemia cells fromalkylating agent induced cell death by three different effects. Eur JMed Res. 2003 Oct. 22; 8(10):438-50. PRECLINICAL TRIALS are initiatedfrom various companies: INOTEK PHARMACEUTICALS, USA,http://www.inotekcorp.com/news/index.htm; Guilford Pharmaceuticals Inc.http://www.guilfordpharm.com/ etc.)

Some additional evidence points towards a crucial role, of PARP1 notonly in neuroprotection and repair, but also in memory formation.Cortical cultures derived from PARP1-knockout mice, or cultures treatedwith a PARP1 inhibitor, are largely resistant to hypoglycaemic neuronaldeath. Very new findings even indicate a role of PARP1 on formation oflong term memories (Suh et al., J. Neurosci. 23 (2003), 10681-10690;Ghen-Ammon et al., Science 304 (2004), 1820-1822).

At present less than 10 PARP-1 inhibitors are in development, althoughnone have yet entered the clinic. Since this class has implications fora variety of serious diseases, most of which represent unmet markets,the further development of molecules such as PJ34 offers considerableclinical and financial promise(http://www.bioportfolio.com/LeadDiscovery/PubMed-030215.htm; Faro R,Toyoda Y, McCully J D, Jagtap P, Szabo E, Virag L, Bianchi C, LevitskyS, Szabo C, Sellke F W. Myocardial protection by PJ34, a novel potentpoly (ADP-ribose) synthetase inhibitor. Ann Thorac Surg. 2002.73:575-81).

However, there appears to be a critical balance of the cell deathpreventing effects of PARP inhibitors, which are mediated by theirability to maintain independently cellular energy metabolism, to inhibitthe activation of endonucleolytic DNA degradation and to prevent cellblebbing and toxic profiles of individual PARP inhibitors.

Overactivation of PARP-1 by severe DNA damage leads to a depletion ofcellular NAD⁺ (i.e., the substrate of PARPs) and ATP (because ATP isrequired for NAD⁺ resynthesis) which may lead to cell necrosis. ie. mildgenotoxic stress activates PARP-1 and stimulates DNA repair, whereassevere damage induces NAD⁺ depletion and necrotic cell death. (Beneke S,Diefenbach J, Burkle A. Poly(ADP-ribosyl)ation inhibitors: promisingdrug candidates for a wide variety of pathophysiologic conditions. Int JCancer. 2004. 111: 813-8). Since the latter is associated withinflammation and pathological tissue damage, modulators of PARP activitycan reduce pathological damage.

A large family of PARP-related proteins with conserved catalytic sitesextends the involvement of enzymatic poly(ADP-ribosyl)ation reactions topotentially many aspects of cell biology, which might ultimately improvepharmacological strategies to enhance both antitumor efficacy and thetreatment of a number of inflammatory and neurodegenerative disorders(Ame J C, Spenlehauer C, de-Murcia G. The PARP superfamily. Bioessays.2004. 26: 882-93.; Beneke et al., 2004 ibid). The involvement ininflammatory processes caused by PARP-related proteins is the subjectmatter of the invention, whereby PARP or related proteins are inhibitedby molecules described herein.

Using a functional cellular model of neuroprotection and a set ofneuronal biomarkers a screening of test compounds for novelneuroprotective modes of action was carried out. Surprisingly, it wasfound that pirenzepine and related compounds have a previously unknownmode of action as PARP inhibitors or PARP binding molecules. Due tothese previously unknown neuroprotective effects, the compounds aresuitable as cytoprotective drugs and new lead structures for thedevelopment and optimization of related compounds with a dual, i.e.M1/PARP1 mode of action, generally for cytoprotection and, particularlyfor the treatment of inflammatory disorders of the lungs.

SIR2 is a protein linked to increased lifespan in yeast and themicroscopic worm Caenorhabditis elegans, potentially delaying thedegeneration of ailing nerve cell branches, relevant for new treatmentsof a wide range of neurodegenerative disorders, including Parkinson'sdisease, Alzheimer's disease, amyotrophic lateral sclerosis (LouGehrig's disease), various kinds of neuropathy, and multiple sclerosis.In mouse nerve cells it has been shown that the protein SIRT1, whichbelongs to a family of proteins known as the SIR2 group, delays thebreakdown of axons in nerve cells mechanically cut off from the cellbody or exposed to a chemotherapeutic agent. Previously evidence wasfound that this process of axonal degeneration may be an activeself-destructive process that the neuron activates under certainconditions. Increased activation of SIRT1 appears to block some or allof those self-destructive processes. Also the possibility of cancerprevention through drugs that increase the activation of SIR2 proteinsis explored (Araki T, Sasaki Y, Milbrandt J. R. Increased nuclear NADbiosynthesis and SIRT1 activation prevent axonal degeneration. Science.2004. 305:1010-3).

There is considerable attention to potential cross-talk between PARP1and SIR2 proteins: PARP-1 is thought to safeguard genomic integrity bylimiting sister chromatid exchanges, with cell death as a consequence ofoverstimulation of PARP-1 by extensive DNA damage. Prolonged PARP-1activation depletes NAD⁺, a substrate, and elevates nicotinamide, aproduct. The decline of NAD⁺ and the rise of nicotinamide maydownregulate the activity the SIR2 NAD⁺-dependent deacetylases, becausedeacetylation by SIR2 is dependent on high concentration of NAD⁺ andinhibited by physiologic level of nicotinamide. The possible linkage ofthe two ancient pathways that mediate broad biological activities mayspell profound evolutionary roles for the conserved PARP-1 and SIR2 genefamilies in multicellular eukaryotes. (Zhang, J. Bioessays, 25 (2003),808-814).

Surprisingly, it was further found that pirenzepine and relatedcompounds have a previously unknown mode of action as SIR2 modulators,e.g. SIR2 binding molecules, with LS-75 (PBD) being a weak SIR 2inhibitor. Due to these previously unknown neuroprotective effects, thecompounds are suitable as cytoprotective drugs, particularlyanti-inflammatory drugs, and new lead structures for the development andoptimization of related compounds with a combined (i.e. M1/PARP1/SIR2)mode of action, generally for cytoprotection and particularly for thetreatment of inflammatory lung disorders.

There is wide acceptance that the similarity between many acuteinfectious diseases, be they viral, bacterial, or parasitic in origin,is caused by the overproduction of inflammatory cytokines initiated whenthe organism interacts with the innate immune system. PARP-1 activity isan established pro-inflammatory mediator in these processes (Clark I A,Alleva L M, Mills A C, Cowden W B. Pathogenesis of malaria andclinically similar conditions. Clin-Microbiol-Rev. 2004. 17: 509-39).For instance, this is relevant for the common lung diseases associatedwith a significant inflammatory component such as severe sepsis, acutelung injury, acute respiratory distress syndrome, and cystic fibrosis.Similar inflammatory processes are involved in allergic lung diseases,such as allergic rhinitis and asthma (Carlsen K H. Therapeuticstrategies for allergic airways diseases. Paediatr-Respir-Rev. 2004. 5:45-51), or in other diseases such as chronic obstructive pulmonarydisease (COPD) of uncertain origin (Hageman G J, Larik I, Pennings H J,Haenen G R, Wouters E F, Bast A. Systemic poly(ADP-ribose) polymerase-1activation, chronic inflammation, and oxidative stress in COPD patients.Free-Radic-Biol-Med. 2003. 15; 35: 140-8. Boulares A H, Zoltoski A J,Sherif Z A, Jolly P, Massaro D, Smulson M E. Gene knockout orpharmacological inhibition of poly(ADP-ribose) polymerase-1 preventslung inflammation in a murine model of asthma. Am J Respir Cell MolBiol. 2003 Mar.; 28(3): 322-9.), or even in cancer progression.

To consider the example of asthma, the disease is characterized by aspecific pattern of inflammation in the airway mucosa. Gene knockouts orpharmacological inhibition of PARP prevent lung inflammation inasthmatic model animals, suggesting that this enzyme is a target for thedevelopment of new therapeutic strategies in the treatment of asthma(Boulares et al. 2003 ibid. Virag L, Bai P, Bak I, Pacher P, Mabley J G,Liaudet L, Bakondi E, Gergely P, Kollai M, Szabo C. Effects ofpoly(ADP-ribose) polymerase inhibition on inflammatory cell migration ina murine model of asthma. Med Sci Monit. 2004 Mar.; 10(3): BR77-83).Despite the present availability of effective and relatively cheaptreatments, approximately 5% of asthmatic patients remain poorlycontrolled, and chronic anti-inflammatory treatment is needed for manypatients. A combination of oral therapy with the present inhaledtreatments might improve this, yet oral therapy presents the problem ofsystemic side effects. Therefore it is desirable that the oral drugshave only minimal adverse effects on normal physiological mechanisms(Barnes P J. New drugs for asthma. Nat Rev Drug Discov. 2004. 3:831-44).

Sepsis is associated with an acquired impairment of the ability of cellsto consume oxygen, a phenomenon called “cytopathic hypoxia,” and this isthought to be mediated, at least in part, by depletion of intracellularlevels of NAD⁺/NADH caused by activation of PARP (Khan A U, Delude R L,Han Y Y, Sappington P L, Han X, Carcillo J A, Fink M P. Liposomal NAD(+)prevents diminished O(2) consumption by immunostimulated Caco-2 cells.Am J Physiol Lung Cell Mol Physiol. 2002 May, 282(5): L1082-91). Indeed,PARP inhibition reduces pathological acute lung injury caused by sepsis(Murakami K, Enkhbaatar P, Shimoda K, Cox R A, Burke A S, Hawkins H K,Traber L D, Schmalstieg F C, Salzman A L, Mabley J G, Komjati K, PacherP, Zsengeller Z, Szabo C, Traber D L. Inhibition of poly (ADP-ribose)polymerase attenuates acute lung injury in an ovine model of sepsis.Shock. 2004 Feb.; 21(2): 126-33).

In cystic fibrosis, virtually all patients become infected withPseudomonas aeruginosa, and such infections, once established, arerarely cleared. Activation of T lymphocytes by factors secreted from P.aeruginosa is thought to produce a lymphocyte-mediated inflammatoryresponse involved in the disease pathogenesis, characterised by heavyinfiltration of the pulmonary epithelium dominated by neutrophils (iepolymorphonuclear cells) leading to airway inflammation and diseasepathology (Bruno T F, Buser D E, Syme R M, Woods D E, Mody C H.Pseudomonas aeruginosa exoenzyme S is a mitogen but not a superantigenfor human T lymphocytes. Infect-Immun. 1998; 66: 3072-9).

In COPD, systemic inflammation is also caused by PARP-1 activation,which contributes to the pathophysiology of COPD patients (Hageman etal., 2003, ibid).

Certain diseases are associated with an increased risk of lung cancer,including idiopathic pulmonary fibrosis, systemic sclerosis, and certainforms of pneumoconiosis. It is well accepted that persistent lunginflammation plays a role in carcinogenesis (Borm P J, Schins R P,Albrecht C. Inhaled particles and lung cancer, part B: paradigms andrisk assessment. Int-J-Cancer. 2004 May 20; 110(1): 3-14. Artinian V,Kvale P A. Cancer and interstitial lung disease. Curr-Opin-Pulm-Med.2004 Sep.; 10(5): 425-34), and it is known that treatment withnon-steroidal anti-inflammatory drugs (NSAIDs) reduces the incidence ofcancers (Gwyn K, Sinicrope F A. Chemoprevention of colorectal cancer. AmJ Gastroenterol. 2002 January; 97(1): 13-21). Furthermore, PARP activityhas been directly associated with inflammation-induced oncogenesis(Martin-Oliva D, O'Valle F, Munoz-Gamez J A, Valenzuela M T, Nunez M I,Aguilar M, Ruiz-de-Almodovar J M, Garcia-del-Moral R, Oliver F J.Crosstalk between PARP-1 and NF-kappaB modulates the promotion of skinneoplasia. Oncogene. 2004 Jul. 8; 23(31): 5275-83).

Patients with rheumatoid arthritis (RA) manifest persistent high levelsof inflammation. Anti-inflammatory reagents belong to the standardtreatments for RA (Furst D E, Breedveld F C, Kalden J R, Smolen J S,Burmester G R, Dougados M, Emery P, Gibofsky A, Kavenaugh A F, KeystoneE C, Klareskog L, Russell A S, van-de-Putte L B, Weisman M H. Updatedconsensus statement on biological agents for the treatment of rheumatoidarthritis and other immune mediated inflammatory diseases (May 2003).Ann-Rheum-Dis. 2003 November; 62 Suppl 2: ii2-9). PARP is also thoughtto be involved in the onset of RA because certain PARP-1 alleles areassociated with susceptiptibility to RA (Pascual M, Lopez-Nevot M A,Caliz R, Ferrer M A, Balsa A, Pascual-Salcedo D, Martin J. Apoly(ADP-ribose) polymerase haplotype spanning the promoter regionconfers susceptibility to rheumatoid arthritis. Arthritis Rheum. 2003March; 48(3): 638-41), and it has been postulated that PARP-1 alleleswere selected by the genetic selective pressure to survive diseaseinfection, because the epidemiology of modern day rheumatoid arthritis(RA) is strikingly similar to the epidemiology of tuberculosis (Mobley JL. Is rheumatoid arthritis a consequence of natural selection forenhanced tuberculosis resistance Med Hypotheses. 2004; 62 (5): 839-43).Thus inhibitors of PARP could contribute to both the treatment ofchronic RA, and its initial prevention. These principles can reasonablybe extended to other autoimmune diseases, such as systemic lupuserythematosus. Indeed, given the widespread incidence of the functionalassociation between PARP activity and inflammation documented here, theprinciples similarly apply to many inflammatory diseases.

Taken together, the above discussion shows that inflammation is involvedin many pathological processes, especially in the lung, and that PARPactivity is important for inflammation. We show that in an experimentalmodel for inflammatory processes, like an LPS challenge of differenttype of cells, e.g. of 3T3 fibroblasts, the compounds have acytoprotective effect. This effect is accompanied by correspondingchanges of apoptotic markers and inflammatory markers, monitored bystaining Western blots with antibodies against PARP-1 and Cox-2.

Thus, a first aspect of the present invention relates to the use of acompound of formula I

wherein A and B are five- or six-membered rings optionally containing atleast one heteroatom selected from N, S and O, wherein the rings areoptionally mono- or polysubstituted with halo, e.g. F, Cl, Br, or I,C₁-C₄-(halo)-alkyl, C₁-C₄-(halo)-alkoxy, amino, C₁-C₄-alkyl-amino, ordi(C₁-C₄-alkyl)amino,W is S, O, NR¹ or CHR¹R1 is hydrogen, Y or COY,R2 is hydrogen or C₁-C₄-(halo)-alkyl, andY is C₁-C₆ (halo)alkyl, or C₃-C₈ cyclo-(halo)-alkyl, wherein the alkylor cycloalkyl group is optionally substituted with a five- orsix-membered ring optionally containing at least one heteroatom selectedfrom N, S and O, and wherein the ring is optionally mono- orpoly-substituted with halo, C₁-C₄-(halo)alkyl, C₁-C₄-(halo)alkoxy,amino, C₁-C₄-alkyl amino, di(C₁-C₄-alkyl)amino or Z,wherein Z is a C₁-C₆ (halo) alkyl group ω-substituted with a groupN(R4)₂,wherein each R4 is independently hydrogen, C₁-C₈ alkyl, orCO—C₁-C₈-alkyl or wherein both R4 together form a five- or six-memberedring optionally containing at least one further heteroatom selected fromN, S and O,wherein the ring is optionally mono- or polysubstituted with halo, C₁-C₄(halo)-alkyl and C₁-C₄(halo) alkoxy,or of a salt or derivative thereof for the manufacture of acytoprotective medicament, particularly an epithelial protectivemedicament for the prevention or treatment of a disease associated withan inflammatory component, e.g. an inflammatory lung disease.

The term “(halo)alkyl” according to the present invention relates to analkyl group which optionally contains at least one halo, e.g. F, Cl, Bror I substituent up to perhalogenation.

The term “salt” preferably refers to pharmaceutically acceptable saltsof compounds of Formula I with suitable cations and/or anions. Examplesof suitable cations are alkaline metal cations such as Li⁺; Na⁺ and K⁺,alkaline earth metal cations such as Mg⁺ and Ca⁺ as well as suitableorganic cations, e.g. ammoniums or substituted ammonium cations.Examples of pharmaceutically acceptable anions are inorganic anions suchas chloride, sulfate, hydrogen sulfate, phosphate or organic cationssuch as acetate, citrate, tartrate, etc.

Derivatives of compounds of Formula I are any molecules which areconverted under physiological conditions to a compound of Formula I,e.g. esters, amides etc. of compounds of Formula I or molecules whichare products of metabolization reactions of a compound of Formula I.

Preferably, the compounds of Formula I are used for the prevention ortreatment of inflammatory or inflammation-associated PARP-1 and/orSIR2-associated disorders, i.e. disorders which are caused by and/oraccompanied by PARP-1 dysfunction, particularly a dysfunctional increasein PARP-1 activity, and/or disorders which are caused by and/oraccompanied by SIR2-dysfunction, particularly a dysfunctional increasein SIR2 activity. For example these disorders include pulmonarydisorders, e.g. lung diseases associated with an inflammatory componentsuch as severe sepsis, acute lung injury, acute respiratory distresssyndrome, cystic fibrosis, asthma, allergic rhinitis, or COPD or lungcancer. Further, these disorders include neuroinflammatory disorders,particularly neuroinflammatory disorders of the central nervous system,e.g. the brain.

A further preferred indication is the prevention or treatment of cysticfibrosis, particularly in persons with impaired function of the cysticfibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel or theprevention or treatment of ulcerative or other inflammatory conditionsof the gastrointestinal system, particularly of persons with impairedfunction of the cystic fibrosis transmembrane conductance regulator(CFTR) Cl⁻ channel.

Still a further preferred indication is for the prevention or treatmentof inflammatory or inflammation-associated processes involved in cancerprevention or progression.

Still a further preferred indication is for the prevention or treatmentof inflammatory or inflammation-associated processes involved inautoimmune diseases such as rheumatoid arthritis or systemic lupuserythematosus, especially for the treatment of patients identified bygenetic or other markers to be more likely to be especially amenable tosuch treatment.

Still a further preferred indication is for the prevention or treatmentof pain, particularly inflammatory or inflammation-associated pain, e.g.chronic pain, since proapoptotic mechanisms play a role in initialphases of various forms of chronic pain (Maione S et al. Apoptotic genesexpression in the lumbar dorsal horn in a model neuropathic pain in rat.Neuroreport 2002 Jan. 21; 13 (1):101-6).

Still, a further preferred indication is for the prevention or treatmentof inflammatory or inflammation-associated ocular disorders,particularly macula degeneration, e.g. moist or dry macula degeneration,glaucoma, e.g. acute, primary, secondary or low angle glaucoma, diabeticretinopathy, anterior or posterior optical neuropathy, retinitispigmentosa, neuritis nervi optici, or central artery obstruction.

For therapeutic applications, the compounds of Formula I may be usedalone or together with other medicaments, e.g. oral asthma medications,clozapine, olanzapine, antidiabetic or anticancer treatments.

In compounds of Formula I, the cyclic groups A and B are preferablyselected from

wherein X is N, CH, or CR₃,V1, V2 or V3 are selected from —O—, —S—, and NR6,R3 is in each case independently halo, C₁-C₄-(halo)-alkyl,C₁-C₄-(halo)-alkyl, C₁-C₄-(halo)-alkoxy, amino, C₁-C₄-alkyl-amino, ordi(C₁-C₄-alkyl)amino,m is an integer of 0-2, andR6 is hydrogen or C₁-C₄-(halo)alkyl.

More preferably, the cyclic group A is selected from

wherein R3 is defined as above,m is an integer of 0-2,r is an integer of 0-1 andR6 is hydrogen or methyl.

More preferably, the cyclic group B is selected from

wherein X, R3 and m are as defined above

In one embodiment, R1 is Y. In this case Y is preferably C₃-C₈cyclo(halo)-alkyl, e.g. cyclopropyl, cyclobutyl or cyclopentyl.

In a further embodiment, R1 is COY and Y is selected from—(CHR7)q-R8wherein R7 is hydrogen, halo or C₁-C₄-(halo)alkyl,q is an integer of 1-4, and preferably 1 andR8 is a five- or six-membered ring optionally containing at least oneheteroatom, wherein the ring is optionally mono- or polysubstituted withC₁-C₄(halo)alkyl or a ω-amino-substituted alkyl group Z as definedabove.

In this embodiment, R8 is preferably selected from

wherein R9 is hydrogen or C₁-C₄(halo)alkyl and R10 is aω-amino-substituted alkyl group Z as defined above.

R9 is preferably a methyl group. The ω-amino-substituted alkyl group Zis preferably a C₁-C₄ (halo)alkyl group having a terminal amino groupwhich is substituted with at least one C₁-C₆ alkyl group, e.g. adiethylamino, or di-isobutylamino group, or with a CO(C₁-C₆) alkyl groupand with hydrogen or a C₁-C₂ alkyl group.

Specific examples of compounds of Formula I are pirenzepine and relatedcompounds as disclosed in FR 1,505,795, U.S. Pat. Nos. 3,406,168,3,660,380, 4,021,557, 4,210,648, 4,213,984, 4,213,985, 4,277,399,4,308,206, 4,317,823, 4,335,250, 4,424,222, 4,424,226, 4,724,236,4,863,920, 5,324,832, 5,620,978, 6,316,423, otenzepad and relatedcompounds as disclosed in U.S. Pat. Nos. 3,406,168, 5,324,832 and5,712,269, AQ-RA741 and related compounds as disclosed in U.S. Pat. Nos.5,716,952, 5,576,436 and 5,324,832, viramune and related compounds asdisclosed in EP-A-0429987, and U.S. Pat. Nos. 5,366,972, 5,705,499, BIBN99 and related compounds as disclosed in U.S. Pat. Nos. 6,022,683 and5,935,781, DIBD, telenzepine and related compounds as disclosed inEP-A-0035519, and U.S. Pat. No. 4,381,301 and salts or derivativesthereof. The above documents are herein incorporated by reference.

Further preferred compounds are 7-azabicyclo-[2.2.1]-heptane and heptenecompounds such as a tiotropium bromide as disclosed in U.S. Pat. Nos.5,817,679, 6,060,473, 6,077,846, 6,117,889, 6,255,490, 6,403,584,6,410,583, 6,537,524, 6,579,889, 6,608,055, 6,627,644, 6,635,658,6,693,202, 6,699,866 and 6,756,392, heterocyclic compounds, e.g.pyrrolidinones, tetrahydropyridines, isoxazocarboxamides, thienopyranecarboxamides, or benzopyranes, such as alvameline tartrate and relatedcompounds disclosed in U.S. Pat. Nos. 6,306,861, 6,365,592, 6,403,594,6,486,163, 6,528,529, 6,680,319, 6,716,857 and 6,759,419,metocloproamide and related compounds as disclosed in U.S. Pat. No.3,177,252 and QNB and related compounds as disclosed in U.S. Pat. No.2,648,667 and salts and derivatives thereof. The above documents areherein incorporated by reference.

Further, the invention encompasses compounds which are metabolized togive diaryl diazepinones according to Formula I such as clozepine andolenzepine.

A further aspect of the present invention relates to the use of acompound which is a dual M1 muscarinic receptor inhibitor and a PARPinhibitor for the manufacture of an airway medicament, preferably forthe prevention or treatment of disorders as indicated above.

The dual inhibitor compound is preferably a moderately strong PARPinhibitor, which has an IC₅₀ value for PARP from 100 to 10000 μM, morepreferably from 250 to 1000 μM. The determination of the IC₅₀ value iscarried out as indicated as in the Examples.

Still, a further aspect of the present invention relates to the use of acompound which is a dual M1 muscarinic receptor inhibitor and a PARPinhibitor and additionally a SIR2 modulator or binding molecule,particularly a SIR2 inhibitor, for the manufacture of a neuro- orcytoprotective medicament, preferably for the prevention or treatment ofdisorders as indicated above.

The compound is preferably a moderately strong PARP inhibitor asindicated above. Further, the compound is preferably a SIR2 inhibitorwhich has a IC₅₀ value for SIR2 from 1 to 10,000 μM, more preferablyfrom 5 to 5,000 μM. The determination of the IC₅₀ value is carried outas indicated in the Examples.

The compounds as indicated above are preferably administered to asubject in need thereof as a pharmaceutical composition, which maycontain pharmaceutically acceptable carriers, diluents and/or adjuvants.The pharmaceutical composition may be administered in the form of atablet, capsule, solution, suspension, aerosol, spray etc. Themedicament may be administered according to any known means, whereinoral, pulmonal and intravenous administration is particularly preferred.The dose of the active ingredient depends on the type and the variety ofdisease and usually is in the range from 1 to 2000 mg/day.

The present application has applications in human and veterinarymedicine, particularly in human medicine.

Furthermore, the present invention shall be explained by the followingFigures and Examples.

FIGURE LEGENDS

FIG. 1: Synthesis of a pirenzepine-related irreversible affinity-tag(11).

FIG. 2: Chemical structures of pirenzepine and its metabolite LS-75(FIG. 2 a); Example of neuroprotective in vitro effect of 1 μMPirenzepine, which prevents neuronal death from chemical ischemia underconditions described (FIG. 2 b). During the course of ischemic insult orrespective rescue by LS-75, concentrations of apoptotic and inflammatorymarkers, PARP-1, Cox-2 and iNOS were quantified by corresponding Westernblots (FIG. 2 c). The survival of neurons in the presence of pirenzepineand LS-75 after challenge with KCN (45 min 3 mM KCN) and β-amyloid (10μM β-amyloid 1-40) is shown (FIGS. 2 d and e). A summary of theseexperiments after three different challenges (excitotoxic, ischemic andβ-amyloid-induced in terms of neuroprotective EC50-values of pirenzepineand LS-75 is shown (FIG. 2 f).

FIG. 3: The silver staining of 1D gels of fractions obtained afteraffinity enrichment is shown in FIG. 3 a: lanes 1-6, 8-17 are controls,lane 7 is the pirenzepine affinity tag enriched material with prominentbands at 113 and 89 kD and a weak band at 110 kD; 3 b: Immunostaining of1D gels of extracts of V56 cells with a specific anti PARP-1 antibody.Lane 16 is an All Blue Marker, 17 is an urea extract and 18 a NP-40extract; lanes 19-22 are eluates from the pirenzepine-affinity column: 3c: The pirenzepine-affinity tag prepared according to the Methodssection irreversibly binds to SIR-2 and provides enrichment of theprotein, as demonstrated by immunostaining 1D gels of extracts of V56cells with a specific anti SIR-2 antibody. Lanes 28 and 39 are molecularweight markers: 29 and 38 are raw extract; 30/31: eluate 1 and flowthrough 1 after overnight incubation of extracts with irreversiblepirenzepine-affinity tag, 32/33: Control, over night incubation of rawextract with streptavidin agarose beads blocked with irreversiblepirenzepine affinity tag; 34/35: Control, over night incubation of rawextract with 5′-AMP-Sepharose beads (Sigma, A3019); 36/37: Control, overnight incubation of raw extract without streptavidin agarose beadscoupled to irreversible pirenzepine-affinity tag.

FIG. 4: Inhibiton of SIR-2 and PARP-1 by pirenzepine and its derivativeLS-75:

In the upper part the corresponding enzymatic activities are plottedagainst increasing concentrations of Pirenzepine and LS-75. As negativecontrols, phenanthridone as a typical PARP-1 inhibitor and nicotineamide as a typical SIR-2 inhibitor were employed (FIG. 4 a). The tablein the lower part of the figure shows respective IC50-values for allsubstances, LS-75 appears to be a moderately strong PARP-1 inhibitor.Pirenzepine is a rather weak PARP-1 inhibitor. Both substances are weakSIR-2 inhibitors (FIG. 4 b).

FIG. 5: Pirenzepine and LS-75 (shown here) protect from LPS challenge(100 ng/ml for 60 min): 5 a protection of 3T3 fibroblasts; 5 bprotection of A 549 cells; 5 c protection of undifferentiated V56embryonic stem cells; 5 d of neurally differentiated V56 embryonic stemcells.

FIG. 6: Effects of Pirenzepine and LS-75 (shown here) are dependent onthe presence of cholesterol-rich lipid rafts. Established methods ofdisruption of these rafts by cholesterol depletion by addition ofmethyl-β-cyclodextrin decreases the neuro protective effect (and alsothe general cytoprotective effect, not shown).

FIG. 7: Organisation and components of neuronal lipid rafts:

These functional membrane compartments are organised by theactivity-dependent interaction of neuregulin (NRG), heparansulfatebinding proteins (HSPG) and dimeric ErbB receptors (ErbB) which regulatethe assembly and activity of a specific set of membrane proteins, whichare essential for some of the most importantneurophysiological/neuropathological processes. Some of them have beenidentified recently as genetic risk factors for Alzheimer's disease(marked AD) and/or schizophrenia (SCH). nAChRa7 is a nicotinicacetylcholine receptor isoform (AD,SCH), NMDAR is an ionotropicglutamate receptor isoform, NRG is a neuregulin (AD,SCH), APP is theamyloid precursor protein (AD), GABA_(A)R is the γ-aminobutyricacid-gated chloride-channel; pTyr stands for phospho-tyrosine; Cho: theraft lipids contain cholesterol (relates to ApoE4, risk factor for AD)and sphingolipids Ex: extracellular; M: membrane compartment; In:intracelluar; Lipid rafts also play a role in non-neuronal cells andmechanisms generally related to inflammation and apoptosis.

FIG. 8: LS-75 prevents poly-ADP-ribosylation under cellular conditions:

The ischemic insult of neural cells by KCN/glucose deprivation induces asubstantial increase in of staining with this antibody, in particular ofa host of proteins in the 100-250 kD range. This effect is reversed byaddition of neuroprotective concentrations of LS-75; here we show thedecrease of poly-ADP-ribosylated proteins during ischemic insult by thepresence of 1 and 10 μM LS-75, respectively. The IC 50 of these effectslies below 1 μM (approx. 0.3 μM).

FIG. 9: Determination of concentrations of Pirenzepine and its two mainmetabolites desmethyl-Pirenzepine (dm-Pirenzepine) and LS-75 (PBD) inplasma and cerebro-spinal fluid (CSF) by HPLC and ultraviolet absorbancedetection; AU are arbitrary units; Pirenzepine and dm-Pirenzepine aredetected at 244 nm; LS-75 (PBD) is detected at 330 nm; clozapine whichabsorbs at both wave lengths is always used as internal standard; Therespective retention times are indicated in minutes next tocorresponding peaks.

FIG. 10: The peak concentrations of Pirenzepine and dm-Pirenzepine arereached about 3 h after oral application of 50 mg Pirenzepine, the leftpart of the graph shows corresponding concentrations in plasma (PLS) andcerebrospinal fluid (CSF) of test rats after 3 h, the right part after 6h, respectively.

FIG. 11: Detection of LS-75 in plasma and CSF of test animals shows thatthe substance passes the blood-brain-barrier (BBB) and is enriched inthe brain after Pirenzepine application. The left part of FIG. 11 showsLS-75 concentrations in plasma (PLS) and CSF after three and six hours:after 6 h there is a substantial increase of LS-75 levels in CSF; in theleft part LS-75 concentrations after application of LS-75 (3 and 6 hlater) are shown: 25-30% of LS-75 pass through the blood-brain-barrier.

FIGS. 12 and 13: Controlled Cortical Impact Injury (CCl)(Craniotomy,metallic piston on dura) trauma associated disorganisation was assessedin terms of protective effects: Markers for cell damage (fast luxol blueand EMAP) reduced by 40-60% in LS-75 treated animals as compared tocontrols, in the contralateral hippocampus.

EXAMPLES Example 1 PARP1 Inhibition

1. Materials and Methods

1.1 Biological Test System: Cell Culture Model for Chemical Ischemia andNeuroprotection

For all experiments, D3 embryonic stem (ES) cells derived from 129/svmice [Okabe et al., 1996] were cultivated for 12 days, with passages ondays 2, 4, 7 and 9 as described previously [Sommer et al., 2004]. Insultconditions: Cells (24-well plates) were pre-incubated with or without 20nM EPO in fresh medium for 24 hours at 37° C. Cells were rinsed oncewith low K⁺ solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.5 mMCaCl₂, 1.2 mM MgSO₄, 11 mM glucose, 15 mM Hepes-NaOH, pH 7.35). Cells(either with or without EPO pre-incubation) were incubated for up to 45min (37° C.) with either low K⁺ solution or with glucose-free low K⁺solution supplemented with 1 mM KCN (chemical ischemia solution [Kume etal., 2002]). Vitality control to asses numbers of surviving neurons wasperformed by a brief stimulation with a low dose of glutamate (10 μM).Afterwards, cells were washed three times with ice cold phosphatebuffered saline (PBS), and then proteins were harvested. Suspended cellswere pelleted at 500×G, and lysed into 9M urea 4% CHAPS. The cell lysatewas desalted with a NAP-10 column (Amersham Biosciences),preequilibrated with the same buffer, and protein content wasdetermined.

1.2 Calcium-Imaging

Functional tests by calcium imaging were performed essentially asdescribed [Sommer et al., 2004]. Briefly, cells were loaded with 2 μM offura-2 μM in DMEM for 45 min at 37° C. in the dark. Measurements ofrelative changes in [Ca²⁺] were made on an inverted epifluorescencemicroscope (Olympus IX70 S1F2) with a Polychrom IV Monochromator(Xe-lamp, USHIO). Excitation wavelengths (λ₁, λ₂) and the emissionwavelength were 340, 380 and 510 nm, respectively. Acquisition andanalysis of data after appropriate stimulation were performed by usingMetaFluor software (Universal Imaging Corporation). Image resolution was168×129 pixels (binning 8×8, pixel size 6.8×6.8 μm). Only cellsidentified as neurons by morphological criteria and occasionalimmunostaining (not shown) and those whose calcium levels returned tothe resting state after the first stimulation were taken into account.Controls included nominal zero calcium (negative) and 5 μM ionomycin(positive), 10 μM glutamate (positive) and depolarisation (55 mM K+)(positive). Pharmacological agents were applied by a multi-valve,single-output focal drug application device (ALA Scientific) with theperfusion system DAD-12. Ratio images were displayed as a percentage ofrelative change in fluorescence over background fluorescence scale forcomparison across experiments [as described in Sommer et al., 2004].During each stimulation event 20 image pairs were acquired.

1.3 Chemical Proteomics: Synthesis of Pirenzepine-Affinity Tag

Pirenzepine was used as a starting structure for the synthesis of anirreversible, i.e. covalently attached, affinity reagent (Fishhook) fortarget proteins. A reactive —SCN group is introduced which binds tolysines in or near the binding site of the compound. A biotinylatedlinker serves for enrichment of bound protein. The synthesis isdescribed in detail co-pending US-application 60/588,354 and 60/620,323which are herein incorporated by reference. In FIG. 1 a synthesis schemeis depcited. The final product, which was used as affinity reagent isthiocyanato11-[2-(4-biocytinyl-piperazin-1-yl)-acetyl]-5,11-dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one,(11).

1.4 Identification and Characterization of Second Binding Site ofPirenzepine

The affinity reagent 11 was used to bind the target covalently fromfractionations of crude cell extracts of D3 ES cells and other celllines, subsequently the affinity purified material was analysed by 1DPAGE, immunostaining, and mass spectrometry.

1.4.1 Fractionation, Isolation, Western Blots, Mass Spectrometry

The subsequent fractionation, isolation and further analysis wasperformed according to published standard procedures (Sommer et al.2004). A commercial anti-PARP antibody was used for staining Westernblots. Mass spectrometry for independent identification ofpirenzepine-tagged proteins was performed as described elsewhererecently (Vogt et al., 2003, Cahill et al., 2003).

Monoclonal anti-PARP antibody was purchased from BD BioScience (Cat# 556362; clone C2-10). Secondary anti-mouse alkaline phosphatase conjugatewas purchased from Sigma (Cat# A9316). NBT/BCIP-westernblot detectionreagents came from Roche Diagnostics (Cat. #1681451), Western LighteningCDP-Star chemiluminescence detection kit was supplied by Perkin Elmer(Cat. #NEL616001 KT). For anti-PARP Western blotting experimentsproteins were separated on 10% polyacryl amide gels and blotted ontonitrocellulose. Blots were blocked with 5% skimmed milk powder in Trisbuffered saline containing 0.1% Tween-20 (TBS-T). Anti-PARP antibody wasincubated over night at 4° C. using a 1:1000 dilution in milk powderTBS-T. Blots were subsequently washed 3 times using TBS-T. Secondantibody was used at a dilution of 1:1000 for NBT/BCIP detection and1:5000 for CDP-Star detection. Gels from various SIR2 containingfractions were blotted onto nitrocellulose membranes and visualizedaccordingly.

For SIR-2 staining the following antibodies were used: primary Ab: A-SiR2 (Upstate, biomol 07-131; Lot:22073); 1:5000 in 5% BSA/1×TBST;secondary Ab: A-Rabbit PE(A-0545) 1:1000 in 5% BSA/1×TBST:

Cox-2 staining was obtained accordingly by using an antibody fromAlexis, (ALX-210-711-1) anti-COX-2 (Cyclooxygenase-2); Rabbit,polyclonal; 1:1000 dilution; secondary antibody was anti-rabbit-AP(Sigma, A3937, 1:1000)

iNOS staining was performed using a polyclonal anti-iNOS, Alexis,1:1000). Blots were washed in TBS/1.0% Tween and incubated with theappropriate secondary antibody-horseradish peroxidase conjugate(anti-rabbit IgG, Sigma, 1:2000).

1.4.2 PARP Inhibition Test

A PARP inhibition assay from R&D Systems was used (Cat. No. TA4669)according to instructions of the supplier.

1.4.3 SIR2 Activity Assay

For measurements of SIR2 activities, the quantitative test kit forNAD-dependent histone deacetylase activity CycLex® SIR2 Assay kit (Cat#CY-1151) was used according to instructions of manufacturer (CycLex Co.,Ltd. 1063-103 Ohara, Tera-Sawaoka Ina, Nagano 396-0002 Japan). Allsubstances tested in the SIR assay were cross-checked for theirinfluence on the lysyl-endopeptidase. For this control an alreadydeacetylated substrate peptide was used in order to measure directlylysyl-endopeptidase activity.

1.4.4 Experimental Model for Inflammation in Neuronal and Non-NeuronalCells

LPS challenge of 3T3 fibroblasts, A 549 cells, V56 embryonic stem cellsand neurally differentiated V56 embryonic stem cells was equallyperformed by exposing cells to 100 ng/ml lipopolysaccharide (LPS, E.coli 0111:B4 LPS from Sigma) for 60 min in the presence or absence ofpirenzepine and related compounds. Cell pellets were furtherinvestigated by Western blot staining with anti Cox-2 and anti iNOSantibodies of 1D polyacrylamide gels.

2. Results

2.1 Neuroprotective Effect of Pirenzepine in Chemical Ischemia

FIG. 2 shows the neuroprotective effects of pirenzepine and LS-75 in thefunctional models outlined in the methods section.

Whereas control cells had a survival rate of 4.8±3.4% (number of cellsat first stimulation: 189 and number of cells at second stimulationafter chemical ischemia, pirenzepine-treated cells had a survival rateof 72.1±4.4% (number of cells at first stimulation: 68 and number ofcells at second stimulation after chemical ischemia: 49) (FIG. 2 b). Inthe lower part (FIG. 2 f) a summary is given for neuroprotective effectsof Pirenzepine and LS-75 in three different functional models: inductionof chemical ischemia as described, induction of excitotoxic cell deathby 100 μM NMDA (or 100 μM HCA as in Sommer et al. 2004) and induction ofneuronal death by 10 μM α-amyloidl-40 (Bachem, Germany); All threechallenges induce an initial calcium overload, which obviously initiatesproapoptotic and proinflammatory events, leading eventually to neuronaldysfunction and cell death. This is shown in FIG. 2 c, by Western blotsof cellular fractions with or without Pirenzepine/LS-75 application,stained for apoptotic markers PARP-1 and inflammatiory marker Cox-2.Additional information on statistics of these experiments are providedin FIGS. 2 d and 2 e.

2.2 Identification of PARP as a Target of Pirenzipine

We then proceeded to synthesize reactive pirenzepine derivatives asshown in FIG. 1; Pirenzepine was used as a starting structure for thesynthesis of an irreversible, i.e. covalently attached, affinity reagentfor target proteins. A reactive —SCN group binds to lysins in or nearthe binding site of the compound. A biotinylated linker serves forenrichment of bound protein. The final affinity reagent,thiocyanato-11-[2-(4-biocytinyl-piperazin-1-yl)-acetyl]-5,11-dihydro-benzo[e]pyrido[3,2-b][1,4]diazepin-6-one(compound (11), FIG. 1), was used to bind the target covalently fromfractionations of crude cell extracts of D3 embryonic stem cells,subsequently the affinity purified material was analysed by 1D PAGE(FIG. 3 a), mass spectrometry and immunostaining. MALDI-TOF analysis ofthe silver stained gels indicated the presence of PARP-1 and SIR-2 inenriched fractions, which was confirmed independently by correspondingstaining of Western blots of 1D gels with a monoclonal anti-PARPantibody (bands at 113 and 89 kD, FIG. 3 b) and a specific antibodyagainst SIR-2 (110 kD, FIG. 3 c).

In the affinity tag incubation 0.5 ml NP 40 stem cell extract (2.3 mgprotein) was incubated with 1 μM affinity tag for 60 min at 37° C. Asurplus of affinity tag was removed by NAP10 gel filtration. Thereaction mixture was bound to streptavidin agarose. Elution occurredwith elution buffer (2% SDS, 62.5 mM Tris-pH 6.8) for 10 min at roomtemperature and 10 min at 95° C. For binding to PARP, a mouse monoclonalantibody (BD Biosciences 1:2000) was used. As detection antibody, ananti-mouse alkaline phosphatase antibody conjugate (1:1000 and NBT/BCIPsubstrate) was used.

2.3 PARP Inhibition Test

Enzymatic tests for SIR-2 and PARP-1 activities, shown in FIG. 4 areveal, that although the affinity tag interacts with both proteins,pirenzepine and LS-75 are PARP-1 inhibitors with IC50-values of 200 and18 μM, respectively and as well appear to be inhibiting SIR-2, but onlyat very high concentrations, with IC50-values beyond 1-5 mM. The tablein FIG. 4 includes controls: nicotine amide had an IC50-value for SIR-2inhibition of approx. 55 μM in our assay, and a typical PARP-1 inhibitorlike phenanthridone had an IC50-value of 7 μM in our assay, which is inagreement with previous reports (North, B. J., Verdin, E. Sirtuins:SIR2-related NAD-dependent protein deacetylases. Genome Biol. 5, 224f,2004; Southan G J, Szabo C. Poly(ADP-ribose) polymerase inhibitors. CurrMed Chem. 2003 Feb.; 10(4):321-40).

Further examples of preferred structurally related compounds suitablefor the present invention are:

-   -   6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (PBD or LS-75), (core        structure, used in PARP1 inhibition test and cell based        neuroprotection assay),    -   Danfenacin hydrobromide (Enablex™, Novartis, M3 muscarinic        antagonists, on market in 2004),    -   Alvameline tartrate (Lu 25-109T, Lundbeck, M1 agonist, M2 & M3        antagonist, disconnected in Phase III of clinical studies since        not efficient in treatment of AD)    -   Impatropium (M1, M2 and M1 antagonist, bronchodilatator)    -   Tiotropium bromide (Spiriva, Boehringer, M1, M2 and M3        antagonist, bronchodilatator, on the market since 2001-2).    -   Metoclopramide, muscarinic antagonist (nonselective one),        dopamine D2 antagonist    -   Telenzepine Dihydrochloride, Sigma,    -   Clozepine,    -   Viramune,    -   Pipenzolate, by Sigma    -   QNB, by Sigma        2.4. General Cytoprotective Effects after an Inflammatory        Challenge (LPS Exposure)

We stimulated 3T3 fibroblasts (FIG. 5 a), A549 cells (FIG. 5 b),undifferentiated V56 embryonic stem cells (FIG. 5 c) and neurallydifferentiated V56 embryonic stem cells (FIG. 5 d with 100 ng/mllipopolysaccharide (E. coli 0111: B4 LPS from Sigma) for 60 min. As aninflammatory marker we again quantified expression of Cox-2 and iNOS byappropriate antibody staining of Western blots of 1D P gels. The resultsshow that Pirenzepine and related substances like LS-75 protect cellsfrom LPS-induced death (FIG. 5 a-d), and ii) that this protective effectis accompanied by a decreased expression of inducible inflammatorymarkers iNOS and Cox-2 (similar to FIG. 2, not shown). Cell survival wasassessed by Trypan Blue staining.

2.5. Influence/Dependence of Effects of Pirenzepine and RelatedSubstances Upon Assembly of Cholesterol-Rich Membrane Domains

Next to the direct effect on PARP-1 and SIR-2 the substances appear tobring about their effects via transient membrane domains,cholesterol-rich lipid rafts, which are thought to be an important in avariety of related signalling pathways (Cuschieri J. Implications oflipid raft disintegration: enhanced anti-inflammatory macrophagephenotype. Surgery. 2004 August; 136 (2):169-75.; Chu C L, Buczek-ThomasJ A, Nugent M A. Heparan sulphate proteoglycans modulate fibroblastgrowth factor-2 binding through a lipid raft-mediated mechanism. BiochemJ. 2004 Apr. 15; 379(Pt 2):331-41; Argyris E G, Acheampong E, Nunnari G,Mukhtar M, Williams K J, Pomerantz R J. Human immunodeficiency virustype 1 enters primary human brain microvascular endothelial cells by amechanism involving cell surface proteoglycans independent of lipidrafts. J Virol. 2003 November; 77(22):12140-51; Nagy P, Vereb G,Sebestyen Z, Horvath G, Lockett S J, Damjanovich S, Park J W, Jovin T M,Szollosi J. Lipid rafts and the local density of ErbB proteins influencethe biological role of homo- and heteroassociations of ErbB2. J CellSci. 2002 Nov. 15; 115(Pt22):4251-62).

In FIG. 6 we show that the neuroprotective effect of Pirenzepine andrelated substances like PBD/LS-75 does not occur in the presence of“raft”-disrupting conditions (FIG. 6 a; β-methyl-cyclodextrin orfilipin) and we conclude, that Pirenzepine and related substances likePBD/LS-75 during do require, at least to some extent, the presence ofcholesterol-rich membrane rafts.

2.6 PARP Inhibition Under Cellular Conditions

A semiquantitative assay for determining PARP inhibition under cellularconditions using a specific antibody against poly-ADP-ribosylatedproteins (primary antibody: anti-poly-(ADP-ribose)-antigen; mouse,Biomol; Cat # SA-216; secondary antibody: anti-mouse, AP; Sigma A9316)was performed. As can be seen in FIG. 8, the ischemic insult of neuralcells by KCN/glucose deprivation (described elswhere in Methodssection), induces a substantial increase in of staining with thisantibody, in particular of a host of proteins in the 100-250 kD range.This effect is reversed by addition of neuroprotective concentrations ofLS-75; here we show the decrease of poly-ADP-ribosylated proteins duringischemic insult by the presence of 1 and 10 μM LS-75, respectively. TheIC 50 of these effects lies below 1 μM (approx. 0.3 μM).

Taken together, in the R & D assay, a histone mix and biotinylated NADand a recombinant monomeric PARP-1 are used; the IC 50 is ˜20 μM. Undercellular conditions PARP-1 poly-ADP-ribosylates a host of nuclearproteins, including topoisomerase 1, 14-3-3 g and PARP-1 itself. Thusunder cellular conditions, the self-modification of PARP-1 anddimerization are regulating its activity, moreover there is a tightinterplay with PARG (poly-ADP-ribosyl-glycohydroxylase).

The quantification of poly-ADP-ribosylated proteins by appropriateWestern blots exactly matches dose-reponse relationship and time framesof the in vitro neuroprotection; we thus conclude that the conditions ofthe R&D assay only partially reflect cellular conditions of PARP-1activity.

2.7 Blood-Brain-Barrier Passage of Pirenzepine and Related Compounds

The blood brain barrier (BBB) passage of Pirenzepine and its metaboliteLS-75 was determined. As already shown in FIG. 6, the neuroprotectiveeffects of e.g. LS-75 during ischemia appears to be dependent on thepresence of (lipid raft-forming) cholesterol, because thecholesterol-depleting agent methyl-b-cyclodextrin preventsneuroprotection. This is in line with the idea that these rafts play acrucial role in underlying signal transduction (see also FIG. 7). Asshown in FIG. 9, we used standard HPLC detection (according to Dusci etal., (2002) J. Chromatogr. B, 773, 191 ff. and Huq et al., (2003)Simplified method development for the extraction of acidic, basic andneutral drugs with a single SPE sorbent-strata X; Phenomenex Inc.Torrance, Calif., USA; Application note SPE/TN-004) to quantifyPirenzepine and its two major metabolites (dm-Pirenzepine and LS-75) inserum and cerebrospinal fluid (CSF) of test animals. For theseexperiments, sets of each 32 rats were given 50 mg/kg Pirenzepine orLS-75 and either killed after 3 h or 6 h, then their plasma and CSF werecollected (128 animals); literature for available information aboutpharmacokinetics and bioavailability of Pirenzepine, underlying therationale of these experiments is e.g.: Jaup and Blomstrand, 1980,Scand. J. Gastroenterol. 66, 35ff.; Homon et al., 1987, Therapeutic DrugMonitoring 9, 236ff.).

Our results show peak concentrations of Pirenzepine and dm-Pirenzepinein plasma of about 2-3 h; in the rat there appears to be virtually nopassage of these two substances into the brain (FIG. 10). In one furtherset of animal experiments we pretreated an identical set of test ratswith Mevastatin, an antibiotic which acts as a potent inhibitor of3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limitingenzyme in cholesterol biosynthesis at a concentration of 0.15 mg/day for14 days, prior to oral gavage of 50 mg Pirenzepine and LS-75. We foundabout 50% higher concentrations of Pirenzepine, dm-Pirenzepine and LS-75in plasma, but far less LS-75 in CSF (data not shown). This is anindication that Pirenzepine partitions into cholesterol-rich parts ofmembranes, which might also be associated with the BBB-passage of LS-75.

2.8 Neuroprotective Effect In Vitro

When LS-75 is applied directly there is a substantial passage throughthe BBB as shown in FIG. 11; the crucial point is that even afterPirenzepine application, but with longer peak times, we observeincreasing amounts of the metabolite LS-75 in the brain. In other words,Pirenzepine serves as a vehicle to transport LS-75, the more activePARP-1 inhibitor into the brain; this is an absolutely novel principle:potentially the muscarinic binding site of Pirenzepine just serves totarget the whole molecule (which is not very anti-PARP-1 active in theperiphery) to cholesterol-rich rafts and subsequently deliver an activepart of the molecule (in this case the metabolite and PARP-1 inhibitorLS-75) into the brain. We claim dual mode of related substances, withone moiety binding to cholesterol-rich rafts or to a raft protein (herea muscarinic receptor), and subsequently enabling passage of BBB for anactive portion of the whole molecule, in this case enriching a PARP-1inhibitor (LS-75) in the brain.

In an in vivo experiment, related to traumatic brain injury (TBI), wecould show a neuroprotective effect for neurons of the contralateralside to trauma. In TBI, very often secondary neurodegenerationcontralateral to the side of injury occurs, probably due tocalcium/glutamate driven proapoptotic mechanisms.

The model employed a direct shock to the dura via a burrhole, animalswere treated according to the following schemes: Vehicle, LS-75, 8animals per group starting the study, 2 spare animals; animals weresacrificed 44 h after injury, the endpoints were survival, post injurysigns, lesion size by FLB (Fast Luxol Blue) and EMAP (Endothelial,Monocyte, Activating Peptide) staining. The dosing i.p. 100 mg/kg 2 hprior to injury; 40 mg/kg i.p. 2 h after injury; 40 mg/kg i.p. 8 h afterinjury; 40 mg/kg i.p. 17 h after injury; 40 mg/kg i.p. 25 h afterinjury; 70 mg/kg i.p. 34 h after injury; Formulation: LS-75 was preparedas a DMSO slurry (not solution) in a mortar and pestle and saline willbe added slowly with grinding to reach 4% DMSO final concentration. Thesuspension is maintained at room temperature and the preparation usedfor the duration of the study. To vary dose, the volume injected maychange. Volume for 100 mg/kg was be 4 mL/kg.

In FIGS. 12 and 13 the corresponding results are shown. 44 h afterinduction of traumatic brain injury (TBI) in an experimental animalmodel, the secondary lesions were reduced by approx. 50% in LS-75treated animals as compared to vehicle controls. EMAP produced clearlabelling of cells at 44 h. EMAP labels were largely associated with theimmediate zone of injury. An analysis was carried out by a “blinded”neurophysiologist who remarked as follows: “EMAP staining in one groupappears to be restricted to the lesion, whereas in another group, it ismore diffuse and associated with vessels”. The diffuse staining was seenin the vehicle group.

The morphological stains HE and Luxol Fast Blue were both useful indisplaying alterations in cells in the contralateral hemisphere. LuxolFast Blue, however, produced a more readily observed staining and sofocussed on it here. An increase in staining by LFB indicates that acell is in transformation and probably reflects the mobilization ofphospholipids and thus neuronal damage.

2.9 Conclusions

Our results clearly show, that pirenzepine and related compounds, inparticular PBD/LS-75 bind to PARP and act as PARP inhibitors.

This property of pirenzepine and related compounds like LS-75 waspreviously unknown and allows the conclusion that pirenzepine andrelated compounds may be used as cytoprotective agents for medicalapplications. Due to the dual mode of action (M1 muscarinic receptor)inhibition and PARP inhibition) these compounds may have superiorproperties over pure PARP inhibitors.

The cytoprotective properties of these and other related compounds arerather due to a hitherto unknown dual mode of action namelymuscarinic/PARP. This novel mixed type of activity can be used for newhigh throughput screening of existing chemical libraries foridentification of novel cytoprotective agents for the treatment ofvarious indications as outlined above.

Generally the invention relates to cytoprotective properties ofcompounds with a dual M1/PARP1 modulating activity for the prevention ortreament of inflammatory disorders.

Example 2 SIR2 Inhibition or Interaction

1. Materials and Methods

1.1 SIR2 Activity Test

For measurements of SIR2 activities, the quantitative test kit forNAD-dependent histone deacetylase activity CycLex® SIR2 Assay kit (Cat#CY-1151) was used according to instructions of manufacturer (CycLex Co.,Ltd. 1063-103 Ohara, Tera-Sawaoka Ina, Nagano 396-0002 Japan).

1.2 Western Blot

Gels from various SIR2 containing fractions were blotted ontonitrocellulose membranes according to standard procedures. Proteins werevisualized using enhanced chemoluminescence (ECL), for Sir 2 stainingthe following antibodies were used: primary Ab: A-SIR 2 (Upstate, biomol07-131; Lot:22073); 1:5000 in 5% BSAr/1×TBST; secondary Ab: A-Rabbit PE(A-0545) 1:1000 in 5% BSA/1×TBST.

2. Results

2.1 SIR2 Interaction with Pirenzepine Affinity Tag, Identification ofSIR2 as a Target of Pirenzipine

FIG. 3 c shows that the pirenzepine-affinity tag prepared according toExample 1 irreversibly binds to SIR2 and provides enrichment of thisadditional target, as demonstrated by immunostaining 1D gels of extractsof V56 cells with a specific antibody. Details are provided in thelegend to FIG. 3.

2.2 SIR 2 Activity Test

Using a raw extract from murine embryonic stem cells as described inSommer et al., (2004) and the commercially available SIR2 activity testdescribed, the following values were recorded in comparison to rawextracts not treated with the drugs.

In FIG. 4 results of a SIR-2 activity test are shown. Pirenzepine andPBD/LS-75 obviously bind to SIR-2 and have a weak inhibitory effect.This opens the route to a corresponding screening for novelstructure/activity relationship studies of related compounds.

2.3 Conclusions

Our results clearly show, that pirenzepine and related structures bindto SIR-2 and can act as weak SIR-2 inhibitors.

This property of pirenzepine and related compounds was previouslyunknown. Due to this mode of action, these compounds may be used ascytoprotective agents and may have superior properties over pure PARPinhibitors.

Thus, the invention also generally relates to cytoprotective propertieswith combined M1/PARP1/SIR2 modulating activity. Moreover the substancesappear to mediate their effects via cholesterol-rich membrane domains,called lipid rafts, as shown in FIG. 6, they thus generally act via ortarget a special assembly of proteins associated with these lipid rafts,like neuregulin, heparanesulfate binding proteins, NMDA receptors,nicotinic receptors, GABA_(A) receptors ErbB receptors and others. Asummary of lipid raft assembly is given in FIG. 7.

Example 3 Cox-2 and iNOS Expression in LPS Challenge and ChemicalIschemia of Neuronal and Non-Neuronal Cells

In the various cellular insult models described here, we always observean initial calcium overload of cells, which subsequently leads toapoptotic cell death, concomitant with increase of apoptotic andproinflammatory markers, such as Cox-2 (see FIG. 2 c and correspondingresults for LPS experiments).

CONCLUSION

The neuro- and more generally cytoprotective effects of Pirenzepine andrelated compounds like PBD/LS-75 on the one hand appear to be mediatedvia PARP-1 and SIR-2 binding and inhibition, and on the other handappear so-called lipid rafts.

A common feature of all the different cellular challenges applied herein the context of said substances is an initial cytotoxic calciumoverload, which subsequently proceeds to inflammatory and apoptoticevents as demonstrated by PARP-1/iNOS/cox-2 staining. Thus the inventionencompasses the use of these substances as treatment in all diseaseindications where calcium overload and inflammatory/apoptotic events arethought to play a major role or potentially are crucial. This includesinflammatory conditions associated with Alzheimer's and Parkinson'sdisease, traumatic brain injury, ALS, multiple sclerosis, migraine andchronic pain syndromes and other non-neuronal diseases as mentionedabove.

OTHER REFERENCES

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1. A method of reducing pathological damage in a subject suffering fromlung cancer, comprising administering to the subject a therapeuticallyeffective amount of a compound of formula I

wherein the cyclic groups A and B are independently selected from

wherein R3 is halo, C₁-C₄-(halo)-alkyl, C₁-C₄-(halo)-alkoxy, amino,C₁-C₄-alkylamino, or di(C₁-C₄-alkyl)amino, m is an integer of 0-2, and Wis NR₁, R1 is hydrogen, Y or COY, R2 is hydrogen or C₁-C₄-(halo)-alkyl,and Y is C₁-C₆ (halo)-alkyl, or C₃-C₈ cyclo-(halo)-alkyl, wherein thealkyl or cycloalkyl group is optionally substituted with a five- orsix-membered ring optionally containing at least one heteroatom selectedfrom N, S and O, wherein the ring is optionally mono- orpoly-substituted with halo, C₁-C₄-(halo)alkyl, C₁-C₄(halo)alkoxy, amino,C₁-C₄-alkyl amino, di (C₁-C₄-alkyl)amino or Z, wherein Z is a C₂-C₆(halo)-alkyl group w-substituted with a group N(R4)₂, wherein each R4 isindependently hydrogen, C₁-C₈ alkyl, or CO—C₁-C₈-alkyl or wherein bothR4 together form a five- or six-membered ring optionally containing atleast one further heteroatom selected from N, S and O, wherein the ringis optionally mono- or polysubstituted with halo, C₁-C₄(halo)-alkyl andC₁-C₄(halo)-alkoxy, a salt thereof.
 2. The method of claim 1, whereinthe cyclic groups A and B are independently selected from

wherein R3 is defined as in claim 1, and m is an integer of 0-2.
 3. Themethod of claim 1 wherein R1 is Y and Y is C₃-C₈-(cyclohalo)alkyl. 4.The method of claim 1 wherein R1 is COY and Y is —(CHR7)_(q)-R8 whereinR7 is hydrogen, halo or C₁-C₄-(halo)alkyl, q is an integer of 1-4, andR8 is a five- or six-membered ring optionally containing at least oneheteroatom, wherein the ring is optionally mono-or polysubstituted withC₁-C₄(halo)alkyl or a ω-amino-substituted alkyl group Z as defined inclaim
 1. 5. The method of claim 4 wherein R8 is selected from

wherein R9 is hydrogen or C₁-C₄(halo)alkyl and R10 is aω-amino-substituted alkyl group Z.
 6. The method of claim 1 wherein thecompound of Formula I is pirenzepine, LS-75, AQ-RA741, viramune, BIBN99, DIBD, or salts thereof.
 7. The method according to claim 1, furthercomprising administering anti-cancer agents with said compound offormula I.